Increasingly, there is a need for proteins which combine two or more functions, such as binding or catalysis, in a single structure. Typically, proteins which combine two or more functions are prepared either as fusion proteins or through chemical conjugation of the component functional domains. Both of these approaches suffer from disadvantages. Genetic "single chain" fusions suffer the disadvantages that (i) only a few (2-3) proteins can be fused (Rock et al., 1992, Prot. Eng. 5, 583-591), (ii) mutual interference between the component domains may hinder folding, and (iii) the size of the fusion protein may make it difficult to prepare. The alternative, chemical cross-linking in vitro following purification of independently expressed proteins, is difficult to control and invariably leads to undefined products and to a severe loss in yield of functional material.
Recently, methods for achieving non-covalent association of two or more of the same functional domains have been developed. This can be achieved through the use of domains attached to peptides which self-associate to form homo-multimers (Pack & Pluckthun, 1992, Biochemistry 31, 1579-1584). For example, the association of two separately expressed scFv antibody fragments by C-terminally fused amphipathic helices in vivo provides homo-dimers of antibody fragments in E. coli (PCT/EP93/00082; Pack et al., 1993, Bio/Technology 11, 1271-1277) or homo-tetramers;(Pack et al., 1995, J. Mol. Biol., 246, 28-34).
To assemble distinct protein functions such as two antibody fragments with different specificities fused to such association domains, the helices must have a tendency to form hetero-multimers. In principle, this could be achieved with complementary helices such as the hetero-dimerizing JUN and FOS zippers of the AP-1 transcription factor (O'Shea et al., 1992, Cell 68, 699-708). The clear disadvantage of association domains based on hetero-associating helices, however, is their pseudo-symmetry and their similar periodicity of hydrophobic and hydrophilic residues. This structural similarity results in a strong tendency to form homo-dimers and, thus, to lower significantly the yield of hetero-dimers (O'Shea et al., 1992, Cell 68, 699-708; Pack, 1994, Ph. D. thesis, Ludwig-Maximilians-Universitat Munchen). Furthermore, the formation of JUN/FOS hetero-dimers is kinetically disfavoured and requires a temperature-dependent unfolding of the kinetically favoured homo-dimers, especially JUN/JUN homo-dimers (PCT/EP93/00082; O'Shea et al., 1992, Cell 68, 699-708; Pack, 1994, Ph. D. thesis, Ludwig-Maximilians-Universitat Munchen). Because of the need for additional purification steps to separate the unwanted homo-dimers from hetero-dimers and the resulting decrease in yield, hetero-association domains based on amphipathic helices do not result in practical advantages compared to conventional chemical coupling.
These disadvantages of the prior art are overcome by the present invention which provides multi-functional polypeptides and methods for the preparation of these multi-functional proteins. This is achieved via the use of association domains which are designed to associate predominantly in a complementary fashion, and not to self-associate.